CN108037279A - A kind of universal ELISA Plate cleaning solution and its application method for improving ELISA kit signal-to-noise ratio - Google Patents
A kind of universal ELISA Plate cleaning solution and its application method for improving ELISA kit signal-to-noise ratio Download PDFInfo
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Abstract
The invention discloses a kind of universal ELISA Plate cleaning solution and its application method for improving ELISA kit signal-to-noise ratio, belong to biological technical field.NaCl of the universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present invention comprising 0.15~0.35M, the Qula of 0.05~0.1%vol lead to the sarcosyl of X 100,3~6g/L, remaining is the phosphate buffer of 0.01M, and wherein the pH value of phosphate buffer is 7.2~7.4.The present invention in cleaning solution by adding the stronger SDS and Triton X100 of washability, the ionic strength of cleaning solution is improved at the same time, effectively control the non-specific interaction between molecule, and then realize the low background and high s/n ratio of ELISA experimental results, improve the sensitiveness and accuracy of detection, and versatility is preferable, there are extremely strong popularization and application potentiality.
Description
Technical field
The invention belongs to biological technical field, and protein in ELISA kit operating process can effectively be controlled by being related to one kind
Between non-specific binding, reduce background, improve the universal ELISA Plate cleaning solution of signal-to-noise ratio.
Background technology
ELISA (enzyme linked immunosorbent assay) is also known as enzyme-linked immunosorbent assay, refers to
Know on antigen or antibody coating and elisa plate, for testing the antibody or anti-that whether has in measuring samples and can specifically bind therewith
It is former.Due to ELISA method have easily and fast, the feature such as sensitivity, be widely used in scientific research and clinical examination.Most
First ELISA method mainly includes direct method and indirect method, as the needs of research, people are basic according to antigen-antibody reaction
Principle, and sandwich method, blocked method etc. have been derived in succession, and have developed corresponding commercial kit.In addition, in order to reduce
The operation sequence of ELISA, people are on the basis of conventional two-step ELISA, by system optimization, and develop " one-step method "
ELISA detection kit.
Although ELISA and deriving method are various, and operating procedure is different.But the core of ELISA is antigen-antibody reaction,
Be between protein molecular interaction (including the organic compound small molecule after modification even metal ion and antibody it is mutual
Effect), in addition to Ag-Ab is specifically bound, also there are non-specific interaction between molecule.And non-specific binding
Background will be increased, and then influence the accuracy of detection.ELISA Plate washing is intended to reduce non-specific binding, reduces background, carries
High s/n ratio, makes testing result more accurate.
For ELISA Plate cleaning solution, it is most classical and be widely used that PBST (i.e. in PBS add 0.05%~0.5%
Polysorbas20 or Tween 80).But find under study for action, ELISA Plate is washed with PBST, is particularly for many ELISA experiments
The antigen or antibody in serum are detected, background is high, and the OD values of negative sample are even as high as 0.2~0.5 (detection serum diluting multiple
Smaller, background is higher), it is difficult to and low concentration detection target is distinguished in measuring samples, reduces the sensitiveness and accuracy of detection.
In addition, being found in research and detection, ELISA detection reagents are used in some commercialization clinic ELISA detection kits or research,
High there are background, its cut off value (decision threshold) is even as high as 0.3~0.4.Therefore, background signal can be reduced by being badly in need of one kind
The universal ELISA Plate cleaning solution of signal-to-noise ratio.
The content of the invention
It is an object of the invention to provide a kind of universal ELISA Plate cleaning solution for improving ELISA kit signal-to-noise ratio, control
Non-specific binding in ELISA experimentations processed between antigen and antibody molecule, can reduce the back of the body caused by uncombined antibody
Scape signal, increases the signal-to-noise ratio of analysis, so as to improve the sensitiveness and accuracy of ELISA detections.
To achieve the above object, technical scheme provides a kind of the general logical of raising TLISA kit signal-to-noise ratio
With type ELISA Plate cleaning solution, the cleaning solution includes NaCl, the triton x-100 of 0.05~0.1%vol of 0.15~0.35M
(Triton-X100), the sarcosyl (SDS) of 3~6g/L, remaining is the phosphate buffer (PB) of 0.01M, wherein
The pH value of phosphate buffer is 7.2~7.4.
Preferably, the 0.01M phosphate buffers are prepared according to the following steps：Take Na2HPO4· 12H2O 3.58g, KCl
0.4g, KH2PO40.54g is dissolved in 1000mL water surely, and pH value is adjusted to 7.2~7.4 (preferable ph with 1M HCl and 1M NaOH
7.4), that is, the phosphate buffer of 1L 0.01M to be made.
Preferably, the content of NaCl is 0.25M in the cleaning solution.
Preferably, the content of triton x-100 is 0.08%vol in the cleaning solution.
Preferably, the content of sarcosyl is 4g/L in the cleaning solution.
The invention further relates to it is a kind of improve ELISA kit signal-to-noise ratio universal ELISA Plate cleaning solution application method,
It the described method comprises the following steps：
(1) antigen or antibody are with after the completion of coated elisa plate reaction, 150 μ L washings are added into the every hole of coated elisa plate
Liquid, drying；
(2) to coated elisa plate per adding 300 μ L cleaning solutions in hole, in washing 3 on the shaking table of 150~200r/min~
5min, drying；
(3) repeat step (2) twice, that is, completes the washing of ELISA Plate, according to ELISA kit design requirement, after progress
Continuous operation.
Preferably, in the step (2) in washing 3min on the shaking table of 180r/min.
The invention has the advantages that：
1st, it is provided by the invention it is a kind of improve ELISA kit signal-to-noise ratio universal ELISA Plate cleaning solution be based on antigen-
Antibody molecule affinity is usually above this feature of affinity between non-specific binding molecule, the addition washing energy in cleaning solution
Power stronger SDS and Triton-X100, while improve the ionic strength of cleaning solution, effectively controls non-specific between molecule
Property interaction, and then realize the low background and high s/n ratio of ELISA experimental results, improve the sensitiveness and accuracy of detection.
2nd, a kind of universal ELISA Plate cleaning solution of raising ELISA kit signal-to-noise ratio of the invention with it is wide in the prior art
The general ELISA Plate cleaning solution PBST used is compared, and can effectively reduce background signal, hence it is evident that improves signal-to-noise ratio；With existing high background
Commercialization or scientific research are compared with the cleaning solution that ELISA kit provides, have higher signal-to-noise ratio, improve detection sensitiveness and
Accuracy；Compared with the cleaning solution that background signal controls good commercialization or scientific research to be provided with ELISA kit, do not influence to examine
Effect is surveyed, is tested suitable for most ELISA, there is extremely strong versatility.
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
The universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment is prepared according to the following steps：
1st, 0.01M PB (pH value 7.4) are configured：
Weigh 3.58g Na2HPO4·12H2O, 0.4g KCl, 0.54g KH2PO4, add 800mL distilled waters to dissolve, adjust
PH value is 7.4, is dissolved to 1000mL.
2nd, ELISA Plate cleaning solution configures：
According to following concentration calculate and weigh prepare 1L cleaning solutions needed for each raw material amount：
The NaCl of 0.25M；
The triton x-100 (Triton-X100) of 0.08%vol；
The sarcosyl (SDS) of 4g/L；
Dissolved with the 0.01M PB of above-mentioned preparation.
The application method of the universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment presses following step
It is rapid to carry out：
(1) antigen or antibody are with after the completion of coated elisa plate reaction, 150 μ L washings are added into the every hole of coated elisa plate
Liquid, drying；
(2) 300 μ L cleaning solutions are added into the every hole of coated elisa plate, in washing 3min on the shaking table of 180r/min, dried；
(3) repeat step (2) twice, that is, completes the washing of ELISA Plate, according to ELISA kit design requirement, after progress
Continuous operation.
Embodiment 2
The universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment is prepared according to the following steps：
1st, 0.01M PB (pH value 7.4) are configured：
Weigh 3.58g Na2HPO4·12H2O, 0.4g KCl, 0.54g KH2PO4, add 800mL distilled waters to dissolve, adjust
PH value is 7.4, is dissolved to 1000mL.
2nd, ELISA Plate cleaning solution configures：
According to following concentration calculate and weigh prepare 1L cleaning solutions needed for each raw material amount：
The NaCl of 0.15M；
The triton x-100 (Triton-X100) of 0.06%vol；
The sarcosyl (SDS) of 6g/L；
Dissolved with the 0.01M PB of above-mentioned preparation.
The application method of the universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment presses following step
It is rapid to carry out：
(1) antigen or antibody are with after the completion of coated elisa plate reaction, 150 μ L washings are added into the every hole of coated elisa plate
Liquid, drying；
(2) 300 μ L cleaning solutions are added into the every hole of coated elisa plate, in washing 4min on the shaking table of 160r/min, dried；
(3) repeat step (2) twice, that is, completes the washing of ELISA Plate, according to ELISA kit design requirement, after progress
Continuous operation.
Embodiment 3
The universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment is prepared according to the following steps：
1st, 0.01M PB (pH value 7.3) are configured：
Weigh 3.58g Na2HPO4·12H2O, 0.4g KCl, 0.54g KH2PO4, add 800mL distilled waters to dissolve, adjust
PH value is 7.3, is dissolved to 1000mL.
2nd, ELISA Plate cleaning solution configures：
According to following concentration calculate and weigh prepare 1L cleaning solutions needed for each raw material amount：
The NaCl of 0.20M；
The triton x-100 (Triton-X100) of 0.09%vol；
The sarcosyl (SDS) of 3g/L；
Dissolved with the 0.01M PB of above-mentioned preparation.
The application method of the universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment presses following step
It is rapid to carry out：
(1) antigen or antibody are with after the completion of coated elisa plate reaction, 150 μ L washings are added into the every hole of coated elisa plate
Liquid, drying；
(2) 300 μ L cleaning solutions are added into the every hole of coated elisa plate, in washing 4min on the shaking table of 190r/min, dried；
(3) repeat step (2) twice, that is, completes the washing of ELISA Plate, according to ELISA kit design requirement, after progress
Continuous operation.
Embodiment 4
The universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment is prepared according to the following steps：
1st, 0.01M PB (pH value 7.2) are configured：
Weigh 3.58g Na2HPO4·12H2O, 0.4g KCl, 0.54g KH2PO4, add 800mL distilled waters to dissolve, adjust
PH value is 7.2, is dissolved to 1000mL.
2nd, ELISA Plate cleaning solution configures：
According to following concentration calculate and weigh prepare 1L cleaning solutions needed for each raw material amount：
The NaCl of 0.30M；
The triton x-100 (Triton-X100) of 0.05%vol；
The sarcosyl (SDS) of 5g/L；
Dissolved with the 0.01M PB of above-mentioned preparation.
The application method of the universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment presses following step
It is rapid to carry out：
(1) antigen or antibody are with after the completion of coated elisa plate reaction, 150 μ L washings are added into the every hole of coated elisa plate
Liquid, drying；
(2) 300 μ L cleaning solutions are added into the every hole of coated elisa plate, in washing 3min on the shaking table of 200r/min, dried；
(3) repeat step (2) twice, that is, completes the washing of ELISA Plate, according to ELISA kit design requirement, after progress
Continuous operation.
Embodiment 5
The universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment is prepared according to the following steps：
1st, 0.01M PB (pH value 7.4) are configured：
Weigh 3.58g Na2HPO4·12H2O, 0.4g KCl, 0.54g KH2PO4, add 800mL distilled waters to dissolve, adjust
PH value is 7.4, is dissolved to 1000mL.
2nd, ELISA Plate cleaning solution configures：
According to following concentration calculate and weigh prepare 1L cleaning solutions needed for each raw material amount：
The NaCl of 0.35M；
The triton x-100 (Triton-X100) of 0.1%vol；
The sarcosyl (SDS) of 4g/L；
Dissolved with the 0.01M PB of above-mentioned preparation.
The application method of the universal ELISA Plate cleaning solution of the raising ELISA kit signal-to-noise ratio of the present embodiment presses following step
It is rapid to carry out：
(1) antigen or antibody are with after the completion of coated elisa plate reaction, 150 μ L washings are added into the every hole of coated elisa plate
Liquid, drying；
(2) 300 μ L cleaning solutions are added into the every hole of coated elisa plate, in washing 5min on the shaking table of 150r/min, dried；
(3) repeat step (2) twice, that is, completes the washing of ELISA Plate, according to ELISA kit design requirement, after progress
Continuous operation.
Checking test 1
The expression with His label recombination chicken interferon-' alpha 's in engineering cell is detected using the ELISA Plate cleaning solution of the present invention
It is horizontal
Concrete operations are as follows：
(1) engineering cell strain for expressing chicken interferon α (with His labels) is inoculated in 24 orifice plates, using free serum culture
Base culture；At the same time using normal Chinese hamster ovary celI as negative control；
(2) when cell covers with more than the 60% of hole, supernatant direct coated elisa plate is taken, per 100 μ L of hole；
(3) 4 DEG C of overnight coated elisa plates discard coating buffer, add the 120 μ L of PBS liquid containing 0.5%BSA at room temperature
Close 30min；
(4) confining liquid is discarded, adds the anti-His monoclonal antibodies of mouse that HRP is marked (to be diluted with the PBS containing 0.5%BSA per hole
500 times) 100 μ L, 37 DEG C of incubation 40min；
(5) ELISA Plate washs：Test group uses the ELISA Plate cleaning solution and washing methods in the embodiment of the present invention 1；Control
Group uses conventional washing liquid (it is 0.1% polysorbas20 to add volume ratio in pH value is 7.4 0.01M PBS), and washing methods is same
Embodiment 1；
(6) develop the color：100 μ L of one-component TMB nitrite ions are added in per hole, 37 DEG C of lucifuges react 15min；
(7) terminate：Terminate liquid (sulfuric acid of 1N) 50 μ L are added in per hole；
(8) OD values measure：OD is read under measurement wavelength 450nm, reference wavelength 620nm.
(9) result：The measured hole and negative control hole OD averages of test group (use cleaning solution of the present invention) be respectively
(1.116 ± 0.05) and (0.056 ± 0.005), signal-to-noise ratio (P/N) are 19.93；And control group (conventional washing liquid (PBST))
OD averages are respectively (1.257 ± 0.096) and (0.245 ± 0.04), and signal-to-noise ratio (P/N) is 5.13.
Checking test 2
B virus of monkey serum antibody is detected using the ELISA Plate cleaning solution of the present invention
Concrete operations are as follows：
(1) the b virus of monkey gD albumen carbonate buffer solution (CBS, pH value 9.6) of Chinese hamster ovary celI recombination expression is diluted
For 1 μ g/mL, elisa plate is coated in, per 100 μ L of hole；
(2) 4 DEG C of overnight elisa plates discard coating buffer, add the 120 μ L of PBS liquid containing 0.5%BSA and close at room temperature
30min；
(3) confining liquid is discarded, 5 times of diluted b virus of monkey positive serum (positive serum B viruses as known to 10 parts are added per hole
Positive serum mixed in equal amounts forms) and negative serum (negative serum B viruses negative serum mixed in equal amounts as known to 10 parts forms)
100 μ L, serum dilution are the PBS containing 0.5% BSA；37 DEG C of incubation 40min；
(4) ELISA Plate washs：Test group uses the ELISA Plate cleaning solution and washing methods in the embodiment of the present invention 1；Control
Group uses conventional washing liquid (it is 0.1% polysorbas20 to add volume ratio in pH value is 7.4 0.01M PBS), and washing methods is same
Embodiment 1；
(5) rabbit-anti monkey IgG secondary antibodies (diluting 20000 times with the PBS containing 0.5%BSA) 100 μ L that HRP is marked are added per hole,
37 DEG C of incubation 40min；
(6) ELISA Plate washs：Same step (4)；
(7) develop the color：100 μ L of one-component TMB nitrite ions are added in per hole, 37 DEG C of lucifuges react 15min；
(8) terminate：Terminate liquid (sulfuric acid of 1N) 50 μ L are added in per hole；
(9) OD values measure：OD is read under measurement wavelength 450nm, reference wavelength 620nm.
(10) result：The measured hole and negative control hole OD averages of test group (use cleaning solution of the present invention) be respectively
(2.250 ± 0.04) and (0.052 ± 0.013), signal-to-noise ratio (P/N) are 43.27；And control group (conventional washing liquid (PBST))
OD averages are respectively (2.406 ± 0.062) and (0.216 ± 0.022), and signal-to-noise ratio (P/N) is 11.39.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (7)
- A kind of 1. universal ELISA Plate cleaning solution for improving ELISA kit signal-to-noise ratio, it is characterised in that：The cleaning solution includes The NaCl of 0.15~0.35M, the triton x-100 of 0.05~0.1%vol, the sarcosyl of 3~6g/L, remaining For the phosphate buffer of 0.01M, the wherein pH value of phosphate buffer is 7.2~7.4.
- 2. the universal ELISA Plate cleaning solution according to claim 1 for improving ELISA kit signal-to-noise ratio, its feature exist In：The 0.01M phosphate buffers are prepared according to the following steps：Take Na2HPO4·12H2O 3.58g, KCl 0.4g, KH2PO4 0.54g is dissolved in 1000mL water surely, with 1M HCl and 1M NaOH adjusting pH value to 7.2~7.4, i.e., the phosphorus of obtained 1L 0.01M Acid buffer.
- 3. the universal ELISA Plate cleaning solution according to claim 1 for improving ELISA kit signal-to-noise ratio, its feature exist In：The content of NaCl is 0.25M in the cleaning solution.
- 4. the universal ELISA Plate cleaning solution according to claim 1 for improving ELISA kit signal-to-noise ratio, its feature exist In：The content of triton x-100 is 0.08%vol in the cleaning solution.
- 5. the universal ELISA Plate cleaning solution according to claim 1 for improving ELISA kit signal-to-noise ratio, its feature exist In：The content of sarcosyl is 4g/L in the cleaning solution.
- A kind of 6. application method for the universal ELISA Plate cleaning solution for improving ELISA kit signal-to-noise ratio, it is characterised in that：It is described Method comprises the following steps：(1) antigen or antibody are with after the completion of coated elisa plate reaction, washing described in 150 μ L is added into the every hole of coated elisa plate Liquid, drying；(2) to coated elisa plate per adding cleaning solution described in 300 μ L in hole, in washing 3 on the shaking table of 150~200r/min~ 5min, drying；(3) repeat step (2) twice, that is, completes the washing of ELISA Plate.
- 7. the application method of the universal ELISA Plate cleaning solution according to claim 6 for improving ELISA kit signal-to-noise ratio, It is characterized in that：In washing 3min on the shaking table of 180r/min in the step (2).
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Cited By (1)
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| CN112946253A (en) * | 2019-12-11 | 2021-06-11 | 广东菲鹏生物有限公司 | Treatment agent for solid phase carrier in immunodiagnostic reagent at closed stage, application and product |
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